Method and apparatus for simultaneous quantitative analysis of several constituents in a sample

ABSTRACT

A method for simultaneous qualitative and quantitative analysis of antigensr antigen-effective substances in a sample provides placing the sample in a carrier medium through which it migrates toward a plurality of physically separate carrier strips. The medium on each strip contains a quantity of a specific ingredient, for example an antibody, which is expected to undergo a specific immunoreaction with one of the constituents in the sample, thereby producing a visible precipitate. The dimensions of the final precipitate then permit a direct measurement of the identified substance. A known concentration of a calibrating substance may be added to the sample to provide for calibration precipitates in the various strips. The apparatus for practicing the method includes a base plate on which the neutral, antibody-free carrier medium as well as the various strips containing media with antibodies or other immunoreactive ingredients are located. An electric field may be applied to aid in the migration of the sample in the medium.

BACKGROUND OF THE INVENTION

The invention relates to a method and an apparatus for carrying out thatmethod for the simultaneous specific and quantitative determination ofseveral substances capable of immunoreaction. The substances areespecially of the kind capable of effective antigen action and thesample migrates from an antibody-free carrier into a carrier containingfurther constituents which, together with the substances to bedetermined, generate an immunoreaction. The substances which are to beanalyzed form precipitates and the extent of migration of the sampletakes place in the direction of an electric field which extends over thesample and over the carrier. An apparatus for carrying out this method,as well as advantageous uses of the method, are also part of theinvention.

An important field of application of a method such as described aboveis, for example, the quantitative determination of proteins in bloodplasma. Such plasma proteins are for example: Prealbumin, Albumin,α-Lipoprotein, α-I-Antitrypsin, α-I-B-Glykoprotein, Gc-Globulin,Coeruloplasmin, α-2-Macroglobulin, Gc-Globulin, Coeruloplasmin,α-2-Macroglobulin, Pseudocholinesterase, hemopexin, Transferrin,β-Lipoprotein, Haptoglobin, Orosomucoid, Antitrypsin, C-reactiveprotein, Fibrinogen, Plasminogen, IgG, IgM, IgA, IgD. The quantitativedetermination of these proteins in many cases permits differentialdiagnostic conclusions to be made or else, when the diagnosis has beenmade, it yields further information regarding the progress and prognosisof a disease (H. H. Marki, Analytische Methoden zur Darstellung derSerumeiweisskorper und ihre Aussagemoglichkeiten fur Klinik und Praxis,Deutsches Medizinisches Journal, 1972 (Jg. 23), S. 317 ff.; H. J. Braun,Immunglobuline, Paraproteine und Blutfarbstoff bindende Proteine undihre Bedeutung fur die Klinik, Deutsches Medizinisches Journal, 1972(Jg. 23) S. 227 ff.; C. O. Kindmark + C. B. Laurell, "Sequential Changesof the Plasma Protein Pattern in Inoculation Hepatitis," Scand. J. clin.Lab. Invest. 29, suppl. 24, 105-115 (1972); J. S., Hepatitis: IgA-Mangelerhoht Risiko, SELECTA 41, S. 3574 (1974); as well as the research of R.Scherer, A. Moratescu, and G. Ruhenstroth-Bauer, "Die spezifischeWirkung der Plasma-Proteine bei der Blutkerperchensenkung").

Several methods have been published for the quantitative determinationof single substances (antigens) which produce immunoreaction withantibodies or, again, methods for the quantitative determination ofsubstances (antibodies) which produce immunoreaction with antigens. Suchmethods are disclosed for example in W. Becker, B. Rapp, H. G. Schwickund K. Storiko, "Methoden zur quantitativen Bestimmung vonPlasmaproteinen durch Immunprazipitation," Zeitschrift fur klinischeChemie und klinische Biochemie, 1968, Heft 3, S. 113-122; W. Becker,"Methoden der qualitativen und quantitativen Immun-Elektrophorese,"Hrsg. Behringwerke AG, Frankfurt, 1972; Prospekt "DAKO-Immunoglobulins"der DAKOPATTS A/S., Danemark, 1972. All these methods are based on theprinciple of immunodiffusion and/or electrophoresis.

When the concentration of antigens is to be determined by the method ofimmunodiffusion, the antigens contained within a sample diffuse into acarrier, for example a layer of agarose gel. The carrier contains only aparticular antibody specific to a particular antigen. When antigens andantibodies make contact within the carrier, they react and form acomplex which is precipitated within the carrier. The diffusion processproceeds until the specific antigen within the sample has been entirelyused up by precipitation with the specific antibody in the carrier. Anyremaining antigens within the sample can continue to diffuse into thecarrier without hindrance and they form no precipitate. The area withinthe carrier in which a precipitate is formed can be made visible bystaining: for example in the so-called radial immunodiffusion process,this area is circular, i.e., it forms a circular zone around theapplication point at which the sample was applied to the carrier. Thedimension of this area in which a precipitate was formed is thus ameasure for the concentration of that antigen within the sample againstwhich the antibody contained within the carrier was specificallydirected.

In principle, this method may be used for a quantitative determinationof all such antigens against which specific antibodies are known oragainst which specific antibodies can be produced. However, this methodis very time-consuming and does not permit the simultaneousdetermination of several antigens.

The above-described method may be accelerated by the use ofelectrophoresis. Use is made of the fact that, under certain ambientconditions, antigens carry an electric charge. If an electric field isapplied in the vicinity of the carrier, the electrically chargedantigens pass through a substantially larger path within the carrierthan would be the case in pure diffusion.

In the general practice, several methods for the determination of theconcentration of individual antigens are used:

a. In the so-called acetate foil electrophoresis a sample (generallyhuman serum) is split up, by an electric field adjacent to the foil,according to the different migration velocities of the individuallycontained components (antigens). After the separation, the sample isstained and is evaluated photometrically. The separation processnormally used in clinical practice only determines a few groups, each ofwhich has the same electrophoretic migration characteristics. Thus thedifferentiation is confined to a determination of α, β, γ-Globulin andAlbumin. Any quantification within these groups, i.e., the determinationof the individual antigens (proteins) contained within each group is notpossible by this method.

b. In the process of radial immunodiffusion, the antigens within asample diffuse from a cylindrical starting orifice in the radialdirection into a layer of agarose gel of uniform thickness whichcontains antibodies. Thus, a cylindrical precipitate is formed aroundthe starting orifice. When the concentration of antibodies in the layerof agarose gel is known, the circular area of the precipitate at thetermination of the diffusion process is a measure of the quantity ofantigen contained in the sample.

This method normally employs immunodiffusion plates having severalstarting orifices. In order to obtain a reference curve, several ofthese starting orifices are filled with different concentrations of astandard solution of known concentration and containing a particularantigen, namely that antigen against which the carrier containsantibodies. The remaining starting orifices are filled with differentsamples whose content of the same antigen is to be determined. When thediffusion process is complete, (after approximately 48 to 72 hours) theareas of the precipitates are measured and the measured values of thedifferent concentrations of the standard solution represent a referencecurve. This reference curve is used to determine the concentration ofthe particular antigens in the sample against which the antibody in thecarrier is directed.

A disadvantage of this method, is first of all, the relatively longduration of the complete formation of the precipitate, i.e., 48 to 72hours. Furthermore, this method is capable of measuring only theconcentration of a single specific antigen at one time. If theconcentration of several specific antigens is to be determined, theabove-described process must be repeated for each antigen. This methodis thus very time and labor consuming.

c. In the so-called rocket immuno-electrophoresis, the disadvantage ofgreat time consumption can be avoided in that the immuno-precipitationis accelerated by the application of an electric field. Just as in theradial immunodiffusion process, individual orifices in a plate, whichhas a surface carrier of agarose gel, are filled with differentconcentrations of a standard preparation so as to obtain a referencecurve, and several samples with unknown content of a particular antigenare then introduced. After the application of an electric field, thesample migrates from the starting orifices into the carrier whichcontains antibodies that are specific against the antigen whoseconcentration is to be measured. For example, if the sample is serum,the antibodies would be specific against a serum protein. the migrationof the proteins in the electric field results in precipitates whichresemble "rockets" whose height is a measure of the concentration ofthose antigens within the sample which have undergone an immunoreactionwith the antibodies contained in the carrier.

It is a disadvantage of this process that, just as in radialimmunodiffusion, there is no possibility for the simultaneousdetermination of several antigens within a probe because the antibodiesin the carrier which form the immunoreaction with the antigens arenecessarily specific for only a particular antigen in the sample. Inorder to determine the concentration of, for example, ten differentantigens within a sample (for example, 10 different plasma proteins in ablood plasma of a patient) 10 different carriers with differentantibodies of the above-described type must be provided with samples andmust be kept in electrophoretic chambers under electric current andsubsequently individually evaluated. Until the present time, the greatamount of instrumentation and apparatus, as well as the cost of labor,has prevented a wide use of this method in clinical practice.

d. In the process of line immuno-electrophoresis (J. Kroll, LineImmunoelectrophoresis, in: M. H. Axelsen et al.: A Manual ofQuantitative Immunoelectrophoresis, Oslo 1973, S. 61-67) a strip of gelwhich contains the sample with different antigens is contacted toseveral gel strips, each of which contains polyvalent antisera. Afterelectrophoresis, the immunoreaction of the different antigens in thesample with the several antibodies contained in the polyvalent antiseraform precipitate lines in each of the gel strips containing antisera.Since the gel strips containing the several polyvalent antisera areadjacent to one another, the precipitate lines in one strip extend intothose of the neighboring strip and thus permit the comparison of theline spectra of the different polyvalent antisera.

e. A known method of the above-described type, on which the presentinvention is based, is the so-called two-dimensionalimmuno-electrophoresis according to Clarke and Freeman. This methodpermits the simultaneous specific (qualitative) and quantitativedetermination of several antigens, i.e., for example several plasmaproteins in blood plasma. In this method, the individual antigens(plasma proteins) are placed in a starting orifice in an agarose gelcontaining no antibodies and are separated purely electrophoretically ina particular direction (first dimension). Subsequently, the electricfield is applied in a second direction (second dimension) which isperpendicular to the first direction. Thus, the once separated antigensnow travel in the direction of the second dimension into a carrier whichcontains antibodies against the various antigens. Thus, in the seconddimension, there takes place the same process ofelectro-immunoprecipitation with the formation of bell-shapedprecipitates which overlap. As before, the area of precipitation is ameasure of the concentration of the particular antigen (plasma protein)in the applied sample. It is not sufficient however, as in rocketelectrophoresis, merely to measure the height; rather the area must bemeasured and this may be done either by planimetry or after transfer ofthe outline to paper of normalized thickness by cutting out the figureand weighing it.

The disadvantage of this known method is the extremely great timeconsumption associated with an evaluation of the precipitates. A furtherdisadvantage is that it is extremely difficult to make a correctinterpretation of the resulting, very complicated appearance of theelectrophoretic picture, i.e., to associate the different areas ofprecipitate with the correct antigens (plasma proteins). This isextremely difficult and requires a great deal of experience,particularly when the electrophoretic picture is substantially differentfrom the expected normal case, as will be true in serum samples takenfrom hospital patients, due to possible pathology.

In summary, the state of the art is such that the determination ofseveral substances capable of an immunoreaction is extremelycomplicated, expensive and time consuming. The methods a, b, c,described above do not permit the simultaneous determination of severalsubstances within a sample. The method d does permit simultaneousdetermination but, as has already been mentioned, has the disadvantagethat the formation of the precipitate areas to be evaluated takes a verylong time and that the evaluation of the precipitates involves a numberof very difficult questions of interpretation and identification,particularly in pathologicaly altered patient sera, while the requireddetermination of the precipitate areas, either by measurement of byweighing, is extremely circuitous and time-consuming. It is thesedisadvantages which are the primary reason that the differentialdiagnostic methods based on the quantitative determination of severaldefined antigens, which are available in preliminary form, have beenused until the present time only in highly specialized laboratories andrequire specially trained operators.

OBJECT AND SUMMARY OF THE INVENTION

It is a principal object of the invention to provide a method for thesimultaneous determination of the type and quantity of substances in abiological sample. The method permits a substantially simpler and fasterevaluation than has heretofore been possible in the methods describedunder subparagraph d above.

It is a further principal object of the invention to provide anapparatus for carrying out the method described by the invention.

These and other objects are attained by the invention by providing thatthe sample is applied to a carrier free from antibodies and migratestherefrom into several different carriers each of which containsdifferent substances which form reactions and precipitates which arespecific, in each case, to the constituents of the sample. The apparatusfor carrying out this method provides a carrier of material free fromantibodies which includes a well or opening for receiving the sample andseveral other carriers each of which contains different substances forforming precipitates with each of the constituents of the sample.

The substances whose presence in the sample is to be determineed by themethod and the apparatus according to this invention are all thosesubstances which form an immunoreaction, i.e., which form precipitateswith other substances, for example antigens forming an immunoreactionwith antibodies or antibodies with antigens. The physical extent of theprecipitates thus formed can be determined, if necessary after staining.Thus, the substances in which the application finds use could also bedescribed encompassingly as "antigen effective" or "antibody effective."In the first case, the individual carriers would include individualtypes of antibodies which are directed against a particular one ofseveral antigens contained in the sample. In the latter case, each ofthe carriers includes an antigen specific to one of the antibodiescontained in the probe. A further important field of application in thefirst named case, as already mentioned, is the determination of aconcentration of individual plasma proteins in blood plasma for purposesof differential diagnosis. In the last mentioned case, a field ofapplication is the diagnosis of autoimmunizing diseases.

The method and apparatus of the invention eliminates the above-describeddisadvantages of the methods and apparatus known to the present state ofthe art in a surprisingly simple manner. It is required only that thesample to be analyzed is supplied into the appropriate well and, after aduration of approximately eight hours, the concentration of severalsubstances can be determined and the number of substances can be quitelarge, up to twenty or more.

During the examination of blood plasma of patients, any deviations ofthe concentration of constituents from a particular nominal value can berecognized in as large a number as desired of diagnostically significantplasma proteins, merely by the inspection of a plate and without greatexpense.

Thus, the invention creates the necessary condition in the laboratoryfor carrying out appropriate serial tests such as are necessary forrecognizing certain pathological, typical deviations of theconcentration of several plasma proteins which could otherwise not evenbe recognized and, if the pathology has been recognized or can bedeveloped, the possibilities for diagnosis could be used in themedicinal practice of physicians not part of a clinical environment.

The invention will be better understood as well as further objects andadvantages thereof become more apparent from the ensuing detailedspecification of two exemplary embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a first exemplary embodiment ofan apparatus which may be used in carying out the method of theinvention in a top view and before use;

FIG. 2 is a section along the line II--II of FIG. 1;

FIG. 3 is a section along the line III--III in FIG. 1;

FIG. 4 is a section along the line IV--IV in FIG. 1;

FIG. 5 is a schematic representation of the apparatus of FIG. 1 after atest on the sample has been performed;

FIG. 6 is a representation of an experiment further explaining theinvention (Experiment No. 1);

FIG. 7 is a schematic representation of the results of a secondexperiment for explaining the invention (Experiment No. 2);

FIG. 8 is a top view of a second exemplary embodiment of the apparatusaccording to the invention after use;

FIG. 9 is a top view of a further exemplary embodiment of the invention;

FIG. 10 is a representation of a photograph of the exemplary embodimentaccording to FIG. 9 in which the individual carriers have beenidentified;

FIG. 11 is a device useful for manufacturing the apparatus according tothe invention; and

FIG. 12 is a top view of the device according to FIG. 10.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Turning now to FIGS. 1-4, there is shown a glass plate 1 on which thereis provided a layer 2 of a width of approximately 1.5 centimetersconsisting of agarose gel, free from antibodies. For the purpose ofapplying this layer 2, the agarose gel is poured onto the glass plate 1in the form of the strip 2. Subsequently, the strip 2 is permitted todry. The strip 2 forms an antibody-free carrier on the glass plate 1.The strip 2 is provided with a recess in the form of a groove or a well3. This may be done by pressing, scraping or the like. The well 3 servesto receive a solution of sample, thus, for example, a blood plasmasample, etc.

Vertically adjacent to the strip 2 are several further strips 12,13, . .. 19. Each of these secondary strips also consists of agarose gel buteach of the vertical strips includes a different and preciselydetermined amount of a specific atibody. Each of the antibodies of eachindividual strip 12, 13 . . . 19 is directed to act against a specificone of those antigens which might be included in the sample contained inthe well 3 and which are to be determined during the examination. If thesample is blood plasma, each of the strips 12, 13 . . . 19 would containantibodies directed against one and only one plasma protein.

It follows that, within each of the strips 12,13 . . . 19, only one ofthe plasma proteins contained in the sample can produce a precipitate byimmunoreaction with the particular antibody to which it is specific. Inthe examination of blood plasma, this means that, in each of the strips,a different plasma protein from the sample in the well 3 forms aprecipitate by immunoreaction with the monospecific antibody containedin that strip.

If the well 3 is filled with a sample, for example blood plasma,containing several antigens (or generally several substances effectiveas antigens) and if an electric field is now applied as indicated inFIG. 1 by the minus signs at the bottom of the strip 2 and the plussigns at the top ends of the strips 12,13, . . . 19, then the individualconstituents of the sample i.e., the individual antigens, migrate withinthe antibody-free agarose gel of the strip 2 in the upward direction asseen in the figure, past the upper edge 2' into the adjacent strips12,13, . . . 19. Since, as already mentioned, these individual stripscontain monospecific antibodies directed against different ones of theantigens within the sample, only a single immunoreaction takes place ineach strip. The precipitated complexes are then redissolved by theexcess antigens. Only when there is no longer an excess of antigens,i.e., when the entire supply of some antigen contained in the sample isused up by immunoreaction against the antibodies contained within one ofthe strips, do the precipitates remain stationary, and thus their limitsbecome recognizable, possibly after staining. Thus there is produced apicture as shown schematically in FIG. 5. In FIG. 5, the variousprecipitates are designated by numerals 12', 13', . . . 19' and areindicated by hatched lines. The limits of the precipitate are designatedwith the numerals 12", 13" . . . 19" and are clearly recognizable.

The position of the limits of precipitation 12", 13" . . . 19", in thedirection in which the antigens migrated from the well 3 into thevarious strips and which is also the direction of the applied electricfield, is a measure of the concentration of the particular antigens inthe blood plasma sample. In particular, the position of the precipitatelimit 12" is a measure of the concentration of that antigen in the bloodplasma sample which formed an immunoreaction with the antibodiescontained in the strip 12. Similarly, the position of the precipitatelimit 13" is a measure of the concentration of the antigen reacting withthe antibody in strip 13, etc.

The plate 1 shown in FIG. 5 is provided with a scale of indicia 4 whichpermits an easy quantification of the position of the precipitatelimits, i.e., their distance a from the upper edge 2' of the strip 2 orfrom the lower edge of the strips 12, 13, . . . 19, respectively.

When human blood plasma is examined, it is possible to so adjust theconcentration of the individual anti-bodies within the strips 12, 13, .. . 19 that when, for example, the blood plasma of a healthy humanpatient is examined, all the precipitate limits would lie at the sameheight, for example along the marker 4'. Thus, when the blood plasma ofa diseased patient is examined under the same conditions, the deviationsof the concentration of particular plasma proteins from the normal valuewould thus be recognizable in an extremely simple manner since theprecipitate limits of those strips which contain antibodies against theparticular plasma proteins would be above or below the marker 4'.Typical patterns of deviation from the normal values could thus beassociated with particular pathological syndromes, which represents aconsiderable progress and substantially facilitates the medicinaldiagnosis. The above-recited statement, namely that the distance a ofthe precipitate limits 12",13", . . . 19" from the upper edge 2' of thestrip 2 or from the lower ends of the strips 12, 13, . . . 19 depends onthe concentration of the antigens which immunoreact with the antibodiesin the strips, has been demonstrated in experiments of which two are nowdescribed.

Experiment No. 1

This experiment is made to show that the distance a of the precipitatelimits from the upper edge 2' of the strip 2 depends on the conentrationof the monospecific antibodies contained in the strips 12, 13, . . . 19against one particular, specific antigen-effective component of thesample. It is further to be shown that, the larger a becomes, the fewerantibodies are present in the strips 12, 13, . . . 19 and vice versa.

The results of the experiment 1 are shown in FIG. 6. The strips 21, 22,. . . 25 of a width of approximately 10 millimeters and a length ofapproximately 72 millimeters each contained antibodies against aparticular plasma protein, namely against α ₂ Macroglobulin, but indifferent concentrations. The strips 21, 22, 23, 24, 25 contained,respectively, 160, 120, 80, 60 and 40 microliters antiserum against α ₂Macroglobulin. The well 103 in the strip 102 was filled withapproximately 200 microliters of a mixture of 300 microliters bloodplasma of a healthy blood donor and 0.7 milliliters agarose. It isnecessary to mix the plasma sample with agarose prior to applicationnecessary in the well 103 so as to guarantee a uniform continuouselectrical conductivity in the agarose gel after filling the well 103with the electrically conductive agarose even after the sample hasmigrated into the strips 21 or 25 or into the strips 12, 13, . . . 19 ofFIG. 5.

The experiment resulted in the following distances a of the precipitatelimits 21", 22" . . . 25" from the upper edge 102' of the strip 102:

    ______________________________________                                        Table of results of                                                           Experiment No. 1                                                                                        Distance of the                                              Microliters Antiserum                                                                          Precipitate                                         Strip No.                                                                              in Strip         (Mean Value) (mm)                                   ______________________________________                                        21       160              7                                                   22       120              9,5                                                 23       80               13                                                  24       60               17,5                                                25       40               24                                                  ______________________________________                                    

This experiment clearly shows that the distance a of the precipitatelimits 21" . . . 25" is a function of the antibody content in the strips21 . . . 25, i.e., the lower the antibody content in this strip, thegreater is the distance a of the precipitate limit from the lower endsof the strips 21, 22 . . . 25 which are adjacent to the upper edge 102'of the strip 102. Conversely, it is also shown that, the larger theantibody content in the strips, the smaller is the distance a.

Experiment No. 2

It is the purpose of experiment No. 2 to demonstrate that the distance aof the precipitate limits from the lower ends of the strips or the upperedge 2' of strip 2 in FIG. 5 depends on the quantity of the appliedsample or, if the sample volume is constant, on the concentration ofthat component within the sample which immunoreacts with the antibodiesin the strips. The results of this experiment are shown in FIG. 7. Thestrips 41 to 44 contain antibodies against α ₂ Macroglobulin in equalconcentration, i.e., each of these strips contained 60 microlitersantiserum against α ₂ Macroglobulin. In order to obtain in each strip animmunoreaction with α ₂ Macroglobulin of different concentrations, fourseparate wells 203', 203", 203'" and 203"" were made in the strip 203.The serum of a healthy blood donor was placed in these wells indifferent concentrations in a solution of agarose. The differentconcentrations of equal volume of sample in the wells are given in thefollowing table. Otherwise, the experimental conditions were the same asthose in Experiment 1. The table also shows the different distances a ofthe precipitate limits from the upper edge 202' of the strip 202.

    ______________________________________                                        Table of Results for                                                          Experiment 2                                                                                  Microliters of Serum                                                                         Distance a of the                                              per 100 Microliters                                                                          Precipitate Limit                              Strip No.                                                                            Well No. of Sample      (Mean value)(mm)                               ______________________________________                                        41     203'     26             11                                             42     203''    36             14                                             43     203'''   46             17                                             44     203''''  56             20                                             ______________________________________                                    

This experiment shows that, for a constant antibody concentration in thestrips 41, 42, 43, 44, the distance a of the precipitate limits is afunction of the applied sample quantity or, if the sample quantity isconstant, it is a function of the concentration of the components in thesample which undergo immunoreaction.

The results of the two experiments, when taken together, show that thedistance a is a function of two parameters, firstly of the antibodyconcentration in the strips 12, 13, . . . 19 and also of the antigenconcentration in the sample. This also implies that, when the antibodyconcentration in the strips 12, 13, . . . 19 is known, then the distancea is a measure of the concentration of the antigen-effective substancein the probe with which the antibodies have produce an immunoreaction.

In order to cause or enhance the migration of the sample placed in thewell 3 into the antibody-containing strips 12, 13, . . . 19, an electricfield is applied to the strips for a duration of at least eight hours(for example overnight). With a suitable electric field strength, afterthis period of time, all antigens have usually completely reacted withtheir corresponding antibodies. Inasmuch as the precipitates in theagarose gel are immovable after a completed reaction, a more prolongedapplication of voltage (for example 24 hours) would not alter theposition of the precipitate limits.

The application of the electric potential may be performed with anycustomary and known electrophoretic apparatus, i.e., in principle by twoelectrodes which are applied via buffer solutions at the locationsindicated in FIG. 1 with plus and minus signs, respectively. Theelectric field strength is approximtely 8 volts per centimeter.

The process which takes place in the individual strips is the formationof a precipitate by immunoreaction under the influence of an electricfield, thus the process may be called an electro-immuno-precipitation.The buffer solution (a barbiturate buffer of pH 8.6 at 0.2 M) is used toinsure a constant buffered pH of 8.6 within the agarose gel. At this pHvalue, the antibody molecules in the strips have neither positive nornegative excess charge and thus do not migrate under the influence ofthe applied electric field as do the individual components of theapplied solution. They form instead a stationary phase into which themobile antigen molecules of the sample migrate.

However, at the preferred pH of 8.6, the blood plasma containsindividual proteins which do not have the negative charge common to mostof the proteins which migrate into the strips under the influence of theapplied field, but are electrically neutral or may even be positivelycharged. Proteins falling into this group are the immunoglobulins Ig G,Ig M, Ig A, Ig D as well as the C-reactive protein and Fibrinogen. Thecharge of these plasma proteins (as well as that of all otherantigen-effective substances without or with reverse charge) can also bechanged chemically for a given pH of 8.6 so as to migrate into thestrips under the influence of the electric field. This may be done by aprior treatment of the sample with formaldehyde. Instead offormaldehyde, the prior treatment may be performed with β-propiolactonor with potassium cyanate (KCNO).

It is also suitable to add to the sample placed in the well 3 a welldefined quantity of a further, foreign antigen-effective substance. Forexample, one may add to a blood plasma sample a particular quantity ofovalbumin, not contained in the blood plasma, to serve as an internalstandard and further to add to all strips 12, 13 . . . 19 a well-definedquantity of antibodies against this substance, i.e., for exampleantibodies against ovalbumin. In that case, there is formed in each ofthe strips 12, 13, . . . 19 a second precipitate whose limit can be usedas a reference, i.e., as a standard magnitude, because it is formedunder the same overall conditions as are the precipitate limits of thesubstances within the sample, and, in addition, the quantity of theantibodies in the strips, as well as the concentration in the sample,will be known in advance. If the internal standard is in hand and theconcentration in the applied sample is also known, then the quantity ofsample no longer affects the quantitative analysis and thus iseliminated as a source of error. In that case, the place of the distancea as described for the exemplary embodiment of FIG. 5 is taken by adistance b between the two precipitate limits within a strip and thisdistance b then provides a measure for the concentration of theantigen-effective substance within a sample, against which theantibodies in that particular strip are effective.

An exemplary embodiment of this type is illustrated in FIG. 8. Shownthere are, in each case, second precipitate limits 12a, 13a, . . . 19awhich refer to the internal standard and which are always at the sameheight, i.e., they are at the same distance from the upper edge 2' ofthe strip 2. In this case, it is the distances b between the precipitatelimits 12" and 12a, etc., which are a measure for the concentration ofthe antigen effect of substances in the sample against which theantibodies contained in the strips 12, 13, . . . 19 are specificallydirected.

The results of two individual determinations of blood plasma proteins ina sample of blood plasma taken with the apparatus of the twoabove-described exemplary embodiments will now be described:

First Exemplary Embodiment

The experiment used a plate 1 on which, as shown in FIG. 1, eight stripswere located. 0.25 ml of serum from a healthy blood donor was mixed with1 ml of a mixture containing 0.4 M formaldehyde in a 0.015 m barbitalbuffer and this final mixture was left standing at room temperature for30 minutes. Subsequently, this mixture was further mixed with 1.250 mlagarose. 0.75 ml of this mixture was placed in the well 3. The strips12, 13, . . . 19 contained monospecific antisera against particularantigens contained within the blood plasma sample whose quantity was tobe determined (compare the table relating to the first exemplaryembodiment). The width of each strip was approximately 10 mm and itslength approximately 74 mm. The quantity of antisera in the strips isgiven in the following table as are the distances a of the precipitatelines from the upper edge 2' of the strip 2 as obtained in theexperiment.

    ______________________________________                                        Table for the First Exemplary Embodiment                                             Quantity and Type of Antiserum                                                                    Distance "a"                                       Strip No.                                                                            contained in the strip (μl)                                                                    (Mean value) (mm)                                  ______________________________________                                        1      100 Anti-IgG        23                                                 2      100 Anti-IgM        2                                                  3      100 Anti-IgA        16,5                                               4       50 Anti-Lipoprotein                                                                              11                                                 5       50 Anti-α.sub.2 -Macroglobulin                                                             11                                                 6      100 Anti-Haptoglobin                                                                              21,5                                               7       50 Anti-Ceruloplasmin                                                                            4,5                                                8      150 Anti-Acidic α-1-Glycoprotein                                                            9                                                  ______________________________________                                    

Second Exemplary Embodiment

0.250 ml of fresh blood plasma from a healthy donor was well mixed with1.0 ml of a solution of formaldehyde (0.4 M formaldehyde in 0.015 Mbarbital buffer) and incubated at room temperature for thirty minutes.Subsequently, 4 ml of agarose was added and a total of 5 ml of thisfinal mixture was placed into the well 3 of a plate as shown in FIG. 9,containing 16 strips 21, 22, . . . 36. The strips 21, 22, . . . 36contained quantities of a monospecific type of antibody against theshown plasma proteins as shown in the following table relating to thesecond exemplary embodiment. An electric potential was applied,resulting in an electric field of 8 volts per cm and, subsequently, thedistance a of the precipitate limits 21", 22", . . . 36" was measured.The following distances (mean values) were obtained.

    ______________________________________                                               Quantity and Type of Antiserum                                                                    Distance a                                         Strip No.                                                                            contained in the strip (μl)                                                                    (Mean value) (mm)                                  ______________________________________                                        21      50 Anti-Fibrinogen 33                                                 22      50 Anti-Plasminogen                                                                              13                                                 23      75 Anti-Orosomucoid                                                                              30                                                 24     100 Anti-Antichymotrypsin                                                                         9                                                  25     150 Anti-α-I-Antitrypsin                                                                    26                                                 26      50 Anti-Ceruloplasmin                                                                            18                                                 27      50 Anti-α-2-Macroglobulin                                                                  17                                                 28     100 Anti-Haptoglobin                                                                              28                                                 29      75 Anti-Hemopexin  12                                                 30     100 Anti-Transferrin                                                                              24                                                 31     100 Anti-α-Lipoprotein                                                                      47                                                 32     100 Anti-β-Lipoprotein                                                                       13                                                 33      50 Anti-β.sub.1 -C/β.sub.1 A Globulin                                                  15                                                 34     100 Anti-IgA        17                                                 35      30 Anti-IgM        19                                                 36     100 Anti-IgG        28                                                 ______________________________________                                    

FIG. 10 is a schematic representation of this type of plate after a testwas made; it was photographed and labeled according to the type ofantiserum used. After staining, the precipitate limits are clearlyrecognizable in the individual strips. It is seen that, for each of theexamined 16 plasma proteins in the second exemplary embodiment, aclearly visible and hence measurable precipitate was formed in theappropriate strip. The plasma proteins which were chosen and shown to bequantifiable simultaneously from the same sample represent those plasmaproteins which have been recognized at the present time to be mostuseful for diagnostic purposes: fibrinogen and plasminogen are decisivefor the diagnosis of blood coagulation disturbances; orosomucoid,antichymotrypsin, antitrypsin and ceruloplasmin are typically increasedin concentration in cases of acute and chronic inflammations as well asin the case of carcinoma; α ₂ -macroglobulin is important in thediagnosis of certain kidney diseases; the simultaneous determination ofhaptoglobin, hemopexin and transferrin permits a differential diagnosisof anemic diseases; α - and especially β -Lipoprotein is important inthe recognition of disturbances in fat metabolism which, in turn,represent a risk factor for the generation of arteriosclerosis; β ₁ C/β₁ A-globulin indicates disturbances in the complementary system; Ig A,Ig M and Ig G are increased or decreased in a typical manner forspecific diseases, especially for infections, allergic diseases, liverdiseases, inflammations and malignant tissue growths. In summary, theresults indicate the wide range of useful possibilities in differentialdiagnosis which is made possible by the invention.

The plates used in the exemplary embodiments, which are provided withthe strips acting as carriers for the antisera, may be produced asfollows: As already described above, antibody-free agarose gel isapplied as a strip to a glass plate. Subsequently, the desired number ofstrips 12, 13, . . . 19 is applied, the exact number corresponding tothe number of antigen-effective substances to be determined. This isalso done by applying warm agarose gel at approximately 50° C containingthe predetermined number of antibodies, by pouring in strips andsubsequent cooling at room temperature, where the gel solidifies. Thedosaging of antibodies in the agarose gel, of which the individualstrips 12, 13, . . . 19 are made, takes place on the basis ofappropriate tests; for example, when blood plasma is examined, thenormal values of the concentration of each blood plasma protein shouldresult in an approximately equally high extent of the precipitate(distance a of the precipitate limit from the lower end of the strip) sothat deviations from the normal values may be easily noticed bydeviations from this straight, normal line which passes through allstrips.

The special advantage of this simultaneous quantitative determination ofseveral antigen-effective substances in a sample, for example of plasmaproteins in blood plasma, is that particular strips in a plate may beinfused with antibodies against exactly those plasma proteins which aresubject to deviations for a particular pathological syndrome (cardiacinfarct, pneumonia, inflammation, tumors, etc.), thus creating animportant tool for differential diagnosis.

In FIGS. 11 and 12, there is shown a relatively simple device forproducing these strips 12, 13 . . . 19 on a plate 1. This deviceconsists of vertically extending plates 50 held together by two rails51. This device is placed so that the ends of the plates 50 are adjacentto the edge 2' of the strip 2. Subsequently, the intermediate spacesbetween the plates 50 are then filled with agarose containing differentmonospecific antibodies, thereby forming the strips 12,13, . . . 19. Theapplication of the electric field does not require special meansbecause, as already explained, all known electrophoretic instruments orsimple devices assembled from elementary electrodes may also be used.

The parallel orientation of the strips as shown in the preferredembodiments is not a necessary requirement, nor do the strips have to benecessarily rectangular. Radial patterns of several strips can also beused. The strips may, in that case, have the shape of circular segmentsproviding that the resulting greater non-linearity of the distance a ofthe precipitate limits from the beginning of the strip can be toleratedin certain special cases of investigation.

As already mentioned, a further variation of the invention is possibleby including in the strips 12, 13, . . . 19 of FIG. 5, not antibodies aspreviously described, but rather antigens, and thus to investigate theindividual components of samples containing antibodies. A field ofapplication of this variant method would be the diagnosis of autoimmunodiseases and allergies. In that case, tissue extracts of the differenttissues, such as heart, kidney, muscle, thyroid gland and nucleic acidswould be incorporated in the strips. The sample to be placed in the well3 would be patient serum. If the patient serum contains autoantibodiesagainst a particular type of tissue, then precipitates will be formed inthe strip containing that particular tissue extract, whereas all otherstrips which contain tissue extracts against which the patient serumdoes not contain autoantibodies would be traversed by the sample withoutforming a precipitate.

What is claimed is:
 1. An apparatus for the simultaneous qualitative andquantitative analysis of a plurality of immuno-reactive substances in asample, comprising:i. a base; ii. a first layer of a first carriermaterial deposited on said base to a defined extent, said first carriermaterial being free from antibodies capable of reacting with anysubstance in said sample; and including a depression for receiving saidsample; iii. a plurality of second layers of a second carrier material,deposited on said base to a defined extent adjacent the extent of saidfirst layer, each of said plurality of second layers including adifferent ingredient capable of undergoing a precipitate-formingimmunoreaction with a specific one of a plurality of substances to beanalyzed in said sample.
 2. An apparatus as defined in claim 1, whereinthe ingredients in said second layers are antibodies effective againstspecific blood plasma proteins.
 3. An apparatus as defined in claim 1,wherein the ingredients in said second layers are extracts of aplurality of body tissues.
 4. An apparatus as defined in claim 1,wherein said extent of said first layer defines an edge and saidplurality of second layers is a plurality of mutually parallel stripseach of which has an end that is contiguous with said edge of said firstlayer.
 5. An apparatus as defined in claim 4, wherein said base is aflat plate.
 6. An apparatus as defined in claim 1, wherein said firstcarrier material and said second carrier material are agarose-gel.
 7. Anapparatus as defined in claim 1, wherein said base is provided with ascale of indicia for measurement of the geometric dimensions of saidprecipitates.
 8. An apparatus as defined in claim 1, wherein saidplurality of second layers includes a further ingredient capable ofundergoing an immunoreaction with a substance added to said sample forpurposes of calibration of said apparatus.